The invention relates to the field of pharmacology and in particular to the screening of chemical substances with potential pharmacological activity using nematode worms such as Caenorhabditis elegans. Specifically, the invention relates to methods adapted for high-throughput screening which are performed in a multi-well plate format.
Caenorhabditis elegans is a nematode worm which occurs naturally in the soil but can be grown easily in the laboratory on nutrient agar or in liquid nutrient broth inoculated with bacteria, preferably E. coli, on which it feeds. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages in its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue types including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of a wild-type worm are relatively easily observed, either directly by microscopy or by using selective staining procedures.
These characteristics of C. elegans make it an extremely useful tool in the drug discovery process. In particular, C. elegans may be used in the development of compound screens, useful in the identification of potential candidate drugs, in which worms are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded.
The possibility that C. elegans might be useful for establishing interactions between external molecules and specific genes by comparison of C. elegans phenotypes which are generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell Biology, Chapter 8, volume 84, Caenorhabditis elegans: Modern Biological analysis of an Organism Ed. Epstein and Shakes, Academic Press, 1995 and J. Ahringer in Curr. Op. in Gen. and Dev. 7, 1997, 410-415.
Rand and Johnson in particular describe compound screening assays in which varying concentrations of the compound to be tested are added to nutrient agar or broth which is subsequently seeded with bacteria and then inoculated with worms. Any phenotypic changes in the worm as a result of exposure to the compound are then observed.
Although the nematode, and in particular C. elegans, is proving a powerful and efficient tool in the identification or discovery of pharmacologically active molecules, the presently known techniques for compound screening do not readily lend themselves to high throughput screening. This is largely because the known assay techniques rely on visual inspection of worms exposed to the compound under test in order to determine whether the compound has an effect on the phenotype of the worms. Consequently, even if an assay were to be performed in the multi-well assay format necessary for high throughput screening it would be necessary to score each individual well by eye in order to determine the outcome of the assay.
There is thus a need for reliable and reproducible screening methods using live C. elegans which do not require scoring by visual inspection and are therefore more suitable for use in automated high throughput screening. The availability of such screening methods would dramatically increase the usefulness of C. elegans as a screening tool, enabling researchers to exploit the enormous potential of C. elegans as a whole animal system for drug discovery and development.
Accordingly, in a first aspect the invention provides a method of identifying chemical substances which have potential pharmacological activity using nematode worms, which method comprises the steps of:
(a) dispensing substantially equal numbers of nematode worms into each of the wells of a multi-well assay plate;
(b) contacting the nematode worms with a sample of a chemical substance;
(c) detecting a signal indicating phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means.
This method is in effect a standard compound screen in which worms are exposed to candidate compounds and changes in the phenotype, behaviour, biochemistry or physiology of the worms as a result of exposure to the compound are recorded. Such assays may be performed using wild-type nematodes, in which case the xe2x80x98changesxe2x80x99 detected in step (c) will generally be changes away from wild type behaviour etc. However, depending on the type of activity to be detected, compound screens can also be carried out using non wild-type worms, for example mutant or transgenic worms which may display non wild-type characteristics. In this case the xe2x80x98changexe2x80x99 detected in part (c) may be a reversion towards wild-type. Typically, compound screening assays involve running a plurality of assay mixtures in parallel with different concentrations of the chemical substance under test. Typically, one of these concentrations serves as a negative control, i.e. zero concentration of test substance. Changes in behaviour, phenotype, biochemistry or physiology etc resulting from exposure to the compound may then be evaluated in comparison to the negative control.
In a second aspect the invention provides a method of determining the mode of action of a chemical substance using nematode worms, which method comprises the steps of:
(a) dispensing substantially equal numbers of a panel of different mutant nematode worms into each of the wells of a multi-well assay plate;
(b) contacting the nematode worms with the chemical substance; and
(c) detecting a signal indicating phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means.
In this method, basic compound screening methodology can be extended to determine the mode of action of a chemical substance. This may be done, for example, by detecting/measuring properties or characteristics of worms exposed to the compound and comparing the result with properties or characteristics of mutant worms carrying mutations in known proteins. Example 4 of the accompanying examples provides an illustration of this in the CNS field.
In a third aspect the invention provides a method of identifying further components of the biochemical pathway on which a compound having a defined effect on nematode worms acts, which method comprises the steps of:
(a) subjecting a population of nematode worms to random mutagenesis;
(b) dispensing one mutagenized F1 nematode worm into each of the wells of a multi-well assay plate;
(c) allowing the F1 nematode worms to generate F2 offspring;
(d) contacting the nematode worms with the compound; and
(e) detecting a signal indicating phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means.
This method of the invention is, in effect, a classic a genetic suppressor screen performed in a multi-well format. In a suppressor screen the aim is to identify a mutation which suppresses the phenotype generated by exposure of the worm to a chemical. Worms carrying suppressor mutations are usually identified on the basis that they exhibit a more xe2x80x98wild typexe2x80x99 phenotype in the presence of the compound, as compared to the phenotype generated by exposure of wild type worms to the same compound. Therefore, to identify a suppressor mutant one effectively looks for mutants which exhibit no or minor changes in phenotypic, physiological, biochemical or behavioural characteristics in part (e) following exposure to the compound.
There are many advantages to be gained from performing genetic suppressor screens in a multi-well format, as described by the inventors. In particular, less compound is required to perform an assay in a multi-well plate, as compared to a standard agar plate assay. Furthermore, as the assay in multi-well plates is performed in liquid, compounds to be tested are taken up more efficiently by the nematodes than in a standard plate assay and also compounds tend to precipitate less in liquid than on agar plates, due to the lower concentration.
In a fourth aspect the invention provides a method of identifying chemical substances which modulate the effect of a first compound, which compound has a defined effect on nematode worms, which method comprises the steps of:
(a) dispensing substantially equal numbers of nematode worms into each of the wells of a multi-well assay plate;
(b) contacting the nematode worms with the first compound;
(c) contacting the nematode worms with a further chemical substance; and
(d) detecting a signal indicating phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means.
This method may be used to screen for antagonists of a given compound. This principle is illustrated in the accompanying Example 8.